PROJECT SUMMARY/ABSTRACT Almost half of all men and women in the United States will be diagnosed with cancer during their lifetimes, resulting in more than 500,000 deaths and an economic burden exceeding $200 billion annually. Cancer immunotherapies, such as vaccines and antibodies, are emerging strategies for specifically targeting malignant cells, while avoiding the potentially dangerous and unpleasant side effects of traditional treatments. Given that cancer is a heterogeneous set of diseases, broadly effective immunotherapeutics would yield substantial public health and economic benefits. Numerous cancers exhibit altered patterns of cell surface carbohydrates. Furthermore, unlike many protein/peptide neoantigens, similar alterations occur across a diversity of malignancies, making tumor-associated carbohydrate antigens an appealing target for immunotherapy development. Unfortunately, carbohydrates are notoriously poor immunogens. As a consequence, despite considerable effort towards realizing the promise of targeting differentially expressed carbohydrate antigens, few therapies or diagnostics have been successfully developed. In those cases where glycans have been successfully targeted, it has been the result of years of singularly focused study or serendipity rather than a successful technological advance to be leveraged. One promising strategy to improve the immune response to carbohydrate targets is to use bacterial outer membrane vesicles (OMVs) as carriers. OMVs, which are composed mainly of periplasmic and outer membrane components of Gram negative bacteria, package multiple copies of a target antigen along with immunostimulatory components in a single, easily purified unit. Glycobia, Inc. specializes in glycoengineering Escherichia coli to produce structurally defined glycoconjugates bearing designer carbohydrates. The central hypothesis of the proposed studies is that glycosylated OMVs (glycOMVs) that display tumor-associated glycans will stimulate a robust immune response that can be subsequently ?glyco-focused? by boosting with the same glycans conjugated to carrier proteins not found on OMVs. Following this heterologous immunization strategy, isolated splenocytes will be used to generate Fab- phage display libraries to identify glycan-directed Fab antibodies that can be converted to full-length monoclonal antibodies for use as direct immunotherapies, or as glycan-targeting sequences in next-generation modalities such as CAR-T, bispecific antibodies, and antibody drug conjugates. The deliverables of this proposal will be a robust method for antibody discovery that elevates and focuses the immune response to otherwise poor immunogens. It is anticipated that this platform will be broadly applicable to the development of engineered vaccine and antibody-based therapies targeting unique carbohydrate antigens that are differentially expressed in human cancers.